Mature-onset cataracts are a major cause of sight impairment and loss in aging populations. The success of cataract surgery and lens implants has ameliorated this for many adults. However, with over 2 million cataract surgeries a year, the $3.6 billion dollar health care costs are a significant burden on the healthcare system. The frequent need for a second procedure, together with occasional other complications, add additional health care costs and burdens. An unfortunate additional problem is that hundreds of thousands of Americans remain unable to access cataract surgery, due to economic, social or psychological factors. Delaying the onset of cataract or slowing their growth - even by a few years - would yield a very significant reduction in health care costs and improvement in the quality of life for senior citizens. This suggests the value of investing in prevention, including the development of small molecule therapies that inhibit cataract formation. Identifying inhibitors of protein aggregation is a major effort for other protein aggregation diseases such as Alzheimer's, Parkinson's and Huntington's. In the last project period, in vitro systems were developed in which partially unfolded human crystallins aggregated into light-scattering complexes at pH7 and amyloid fibers at low pH. The in vitro aggregation reactions capture a number of the properties of cataract formation within the lens. This project will take advantage of these experimental systems by performing high-throughput screening for compounds that inhibit the in vitro initiation or propagation of crystallin aggregation reactions. The initial effort will tap the 250,000 compounds available through the Harvard Medical School Longwood Facility, and a smaller set of active amyloid inhibitors synthesized in the MIT Chemistry Department. The target reactions employ human 3D- and 3C-crystallins carrying amino acid modifications representing various forms of oxidative and mutational damage that have been found in cataracts. In addition, fragments of human cataractous aggregates from surgical procedures will be used as seeds in the in vitro reaction, and screened for compounds that inhibit addition of crystallin chains to the seeds. The initial hits will be characterized as to whether they inhibit the initiation or propagation of the aggregation process, and also by their effects on substrate binding by the lens chaperone 1-crystallin. To improve the search for potential target binding sites and putative hits, NMR and cryoEM resources will be tapped to characterize the crystallin substrate/inhibitor interactions and the conformation of substrate/chaperone binding sites. This will enable utilization of powerful in silico computational searches for target sites and new compounds. The Initial hits will be improved by chemical modification and further characterization in vitro. Such putative anti-cataract agents would then be candidates for testing in animal models of cataract. PUBLIC HEALTH RELEVANCE: Cataracts are the leading cause of vision loss among older Americans. The proposed identification of a set of small molecules that interfere with aggregation of damaged or perturbed human lens crystallins in the test tube will provide candidates for low-cost anti-cataract therapy. If a subset of such agents were able to delay the onset of cataract by a few years, or retard cataract growth, they would significantly slow vision loss and the need for expensive cataract surgery for many older adults.